Field of the Invention
The present invention relates to a heparosan-producing bacterium and a method for producing heparosan.
Brief Description of the Related Art
Heparosan (also referred to as N-acetylheparosan) is a polysaccharide constituted by a repetition structure of a disaccharide having a glucuronic acid (GlcUA) residue and an N-acetyl-D-glucosamine (GlcNAc) residue [→4)-β-GlcUA-(1→4)-α-GlcNAc-(1→].
In nature, heparosan is produced by the Escherichia coli K5 strain and the Pasteurella multocida type D strain as a capsular polysaccharide (Lindahl U. et al. (1998) J. Biol. Chem., 273(39):24979-24982). These heparosan-producing bacteria are pathogenic and cause urinary tract infections, atrophic rhinitis, etc. in mammals.
In the Escherichia coli K5 strain, two kinds of glucosyltransferases, which are heparosan synthetases, and six kinds of heparosan efflux carriers are required for the biosynthesis of heparosan. That is, GlcNAc and GlcUA are first alternately added to a non-reducing end of the sugar chain by the glucosyltransferases (KfiA and KfiC), and the heparosan chain is thereby extended (Hodson N. et al. (2000) J. Biol. Chem., 275(35):27311-27315). Then, the heparosan chain is transported to the cell surface by the heparosan efflux carriers, which include KpsC, KpsD, KpsE, KpsM, KpsS, and KpsT (McNulty C. et al. (2006) Mol. Microbiol., 59(3):907-22). It is thought that the heparosan chain is fixed to a phosphatidic acid molecule in the outer membrane of Escherichia coli on the cell surface through lipid substitution at the reducing end (Jann B., Jann K. (1990) Curr. Top Microbiol. Immunol., 150:19-42).
In the Escherichia coli K5 strain, the heparosan synthetase genes and the heparosan efflux carrier genes form a cluster on the chromosome. The cluster is divided into regions 1 to 3, and region 2, located at the center of the cluster, encodes the four proteins including the heparosan synthetases, KfiA, KfiB, KfiC, and KfiD.
The Pasteurella multocida type D strain has PmHS1, which acts as a heparosan synthetase (glucosyltransferase) (Kane T. A. et al. (2006) J. Biol. Chem., November 3; 281(44):33192-33197). PmHS1 has active domains homologous to both KfiA and KfiC of the Escherichia coli K5 strain, and it catalyzes a polymerization reaction using both UDP-glucuronic acid and UDP-N-acetylglucosamine as substrates. However, to date, no heparosan efflux carriers of the Pasteurella multocida type D strain have been eluciated.
Heparin is one of anticoagulants, and is useful in therapeutic treatments of thromboembolism and disseminated intravascular coagulation (DIC), prevention of blood coagulation during artificial dialysis and extracorporeal circulation, and so forth. Heparosan is a sugar chain structure of heparin, and can be converted into a heparin-like polysaccharide through such steps as deacetylation, epimerization, sulfation, and molecular weight adjustment (Lindahl U. et al. (2005) J. Med. Chem., 48(2):349-352 and Zhang Z. et al. (2008) Journal of the American Chemical Society, 130(39):12998-13007).
Heparin exhibits an anticoagulant activity through activation of antithrombin III, which is an anticoagulant. Antithrombin III binds to the active serine moieties of thrombin, Xa factor (active type of X factor), and other serine proteases to inhibit them. Thrombin is a blood coagulation factor, and the Xa factor is a factor involved in the maturation of thrombin. Heparin binds to this antithrombin III to change the structure thereof, and thereby activates the inhibitory activity. Thrombin shows higher affinity for the heparin-antithrombin-III complex compared with the Xa factor.
Low molecular weight heparins having an average molecular weight of 4000 to 6000 Da, which are obtainable by enzymatic or chemical treatments of heparin and fractionation, show less adverse reaction of hemorrhage, and frequency of use thereof is increasing in recent years. Since the low molecular weight heparins have a short sugar chain length, they can barely bind with thrombin, although they can bind with antithrombin III. For the inhibition of thrombin by the heparin-antithrombin III complex, binding of thrombin to heparin is necessary, but for the inhibition of the Xa factor by the heparin-antithrombin III complex, binding of the Xa factor to heparin is unnecessary. Therefore, the low molecular weight heparins hardly inhibit the activity of thrombin, but can inhibit the activity of the Xa factor.
Most of the currently available heparin preparations utilize extracts of porcine intestinal mucosa. However, in 2008, a fatal accident occurred as a result of contamination of impurities, and therefore the production and development of quality-controlled non-animal heparin was investigated.
It has recently been demonstrated through laboratory scale research that heparosan obtained from the Escherichia coli K5 strain can be enzymatically converted into a heparin-like anticoagulant polysaccharide (Lindahl U. et al. (2005) J. Med. Chem., 48(2):349-352 and Zhang Z. et al. (2008) Journal of the American Chemical Society, 130(39):12998-13007). Furthermore, heparosan can also be utilized for uses other than heparin manufacture (WO2009/014559).
Large scale production of heparosan using Escherichia coli K5 is being investigated, and it has been reported that 15 g/L of heparosan was produced in a 7-L fermentation tank (Wang Z. et al. (2010) Biotechnol. Bioeng., 107(6):964-973, Japanese Patent Laid-open (Kohyo) No. 2013-503606). In order to supply heparosan on an industrial scale as a raw material of heparin production, it must be scaled up to the order of 100,000 L, but there are issues that must be resolved, particularly concerning improving the substrate consumption rate, increasing the oxygen supply in fermentation tank, etc.
Furthermore, a heparosan-producing bacterium produced from a nonpathogenic Escherichia coli BL21(DE3) host has very recently been reported (Zang C. et al. (2012) Metabolic Engineering, 14(5):521-527). That is, in flask culture of the BL21 strain that had been introduced with an expression vector pETDuet-1 carrying the four heparosan biosynthesis genes, kfiA, kfiB, kfiC, and kfiD, which constitute region 2 of the Escherichia coli K5 strain, production of 334 mg/L of heparosan was confirmed.
Although the factors required for the heparosan production have been elucidated, factors that improve heparosan-producing ability of a heparosan-producing bacterium have not been previously reported.